Molecular basis of replication origin activation/silencing during tissue-specific gene regulation. Understanding long-range regulation of gene expression is of paramount importance to comprehend the functional organization of mammalian genomes, as well as cell differentiation and development. This could also help to improve the design of transgenic vectors and to better appreciate the effect of chromosomal deletions, insertions, and translocations on the expression of adjacent genes. We have recently shown that regulation of gene expression in the immunoglobulin heavy chain (Igh) locus is accompanied, during B cell development, by long range changes in replication origin activity. The activity of multiple origins is modified across genomic regions that are hundreds of kilobases in size. This is one of the earliest modifications detectable during the activation of the Igh locus. Elucidating the mechanisms involved in origin regulation during cell differentiation will help to clarify how tissue-specific patterns of gene expression are established and maintained in proliferating cells. Preliminary results indicate that the deletion of a previously characterized long range regulatory element, the Igh intronic enhancer, reduces gene expression from various developmentally regulated promoters but does not prevent the activation of the developmentally regulated origins of replication. The accessibility of the same genomic region to other B cell-specific events (e.g. D-J recombination) is also not significantly affected. This suggests that the long range regulation of origin activity and locus accessibility are at least partially independent from enhancer elements. Clusters of origins active in early S phase could characterize genomic regions that become accessible to transcriptional regulation or recombination during cell differentiation. The identification of such clusters at various B cell-specific gene loci will allow us to identify functional units of mammalian chromosomes. Preliminary results also indicate that developmental regulators, such as Pax5, are directly or indirectly involved in origin regulation across large portions of the Igh locus. We will examine primary wild-type and Pax5 -/- pro-B cells (AIM I), as well as Pax5 reconstituted pro-B cells (AIM II), and pro-B cells in which Pax5 has been conditionally inactivated (AIM II), to clarify the relationship between developmental regulation of gene expression and origin activation at various gene loci. We will also begin to dissect the pathway by which Pax5 or other developmental regulators induce these changes (AIM III).